Molecular Biology of Wilm's Tumour.

at an early age. Furthermore, a genetic predisposition to develop the tumour is associated with aniridia, genitourinary abnormalities and mental retardation (the WAGR syndrome) (1). Children with this rare syndrome typically carry a germline deletion involving band p13 on one of the two (parentally-derived) chromosome 11 homologues (2). The inherited 11 p deletion in WAGR and hereditary Wilms' patients is thought to represent the first of two events required for initiation of Wilms'


INTRODUCTION
Wilms' Tumour (Nephroblastoma) is an embryonal tumour of the kidney, which affects approximately 1 in 10,000 children and accounts for 6% of all paediatric cancers. Although the vast majority of Wilms' tumours are sporadic, in the small percentage of hereditary cases (4-8%) observed, the tumour is often bilateral and arises at an early age. Furthermore, a genetic predisposition to develop the tumour is associated with aniridia, genitourinary abnormalities and mental retardation (the WAGR syndrome) (1). Children with this rare syndrome typically carry a germline deletion involving band p13 on one of the two (parentally-derived) chromosome 11 homologues (2). The inherited 11 p deletion in WAGR and hereditary Wilms' patients is thought to represent the first of two events required for initiation of Wilms' tumour, as postulated by Knudson from epidemiological studies (3). In addition, the specific loss of chromosome lip alleles has been shown in sporadic Wilms' tumours (4)(5)(6)(7). This loss of normal cellular lip sequences in Wilms' tumourigenesis, correlates with the principle of development of somatic homozygosity of a recessive defect within 11 pi3. It has consequently been postulated that the intact wild-type Wilms' tumour locus at 11 pi3 may encode a tumour suppressor and/or differentiation function (8). Interestingly, the same pathogenetic mechanism involving the 11 pi3 locus is implicated in other closely related childhood tumours: hepatoblastoma, rhabdomyosarcoma and adrenal carcinoma (8).
This study concerns an analysis of Wilms' tumour, adjacent normal kidney and blood cell DNA from 6 sporadic Wilms' tumour patients, for any chromosome 11 gene changes which might be related to tumour development.

METHODS
Subjects: The six patients studied were between 2.5 and 6 years old with unilateral sporadic Wilms' tumour and no evidence of aniridia. Tumour and adjacent nontumour kidney tissue were obtained at tumour resection, prior to any chemotherapy or radiotherapy. In all the specimens examined from each patient, this neighbouring non-tumour tissue was histologically normal. Small pieces of fresh tumour and adjacent kidney were explanted on to mitomycin C treated 3T3 feeder cell monolayers and any cell cultures obtained were maintained for up to 9 passages with feeders and Dulbecco's modification of Eagle's medium supplemented with 15% fetal bovine serum, hydrocortisone (1 ug/ml), insulin (0.2 units/ml), and epidermal growth factor (10ug/ml) (9).
Where possible blood samples were also obtained and the leukocytes transformed with Epstein-Barr virus (EBV) to establish lymphoblastoid B cell lines as a permanent source of constitutional DNA (10). Post-hybridization washing and autoradiography. Hybri' dized blots were washed in 2xSSC + 0.5% sodiu^ dodecyl sulphate (SDS) for 2 hours at 65?C, and then exposed to X-ray film between intensifying screens at -70?C for 1-3 days. Autoradiographs were analysed bV scanning densitometry (Bio-Rad model 620, video den' sitometer) to allow quantitative comparison between Wilms' tumour and normal kidney/B cell DNA.

RESULTS AND DISCUSSION
? If DNA is digested with appropriate restriction enzym^ (which cleave the DNA at specific short sequences) an Southern blot analysis performed with chromosome 1 DNA probes, it is possible to distinguish the materna paternal chromosome homologues (alleles) of a gene t>V means of restriction fragment length polymorphisms (RFLPs). There are natural variations in base sequence (,n and around genes) between individuals, due to po'n mutations or to the presence/deletion of short repetitive sequences. These natural variations in base sequent will generate changes in the length of the restrict!011 fragment on which the gene of interest is locate0' ain only one copy of a gene (one allele): hemizygosr ' 0r that the tumour cells have lost one allele and aj ^Plicated the remaining allele to give 2 copies of one homozygosity). In fact, in 3 of the 6 patients led we have been able to detect this loss of heterozy-SuSltY' w'th at least one chromosome 11 p gene probe (as ^rnarized in Table 1 and partially illustrated in Fig. 1). Fig. 1 (a) and (b) clearly illustrates the development of hemizygosity for both calcitonin and c-Ha-ras-1 respectively, in patient 3 Wilms tumour DNA. One polymorphic band is lost in the tumour compared to normal kidney DNA, but there is no simultaneous duplication of remaining allele. Development of homozygosity for c-Ha-ras-1 in Patient 3 Wilms' tumour is similarly illustrated in Fig. 1 (b), where scanning densitometry revealed that the remaining allele is reduplicated in the Wilms' tumour.
In  Fig. 2 (c), (d)). Furthermore, one renal cell culture derived from one of these two patients (patient 1) showed an even greater degree of amplification (up to 30x) for both the 11 pi5 genes: B-globin and c-Ha-ras-1 (see Fig. 2 (c) and (e)). A further normal kidney cell culture derived from a third patient (patient 6) showed 3 x amplification of calcitonin, another 11 p15 gene (data not shown). In the cultures these amplifications were associated with polymorphic changes (e.g. see Fig. 2 (c) and (e)). Importantly, no amplification was observed in B cell DNA from these patients. Neither were they widespread random alterations, because DNA markers examined on other chromosomes by re-probing the same DNA digests showed normal dosage (e.g. with Ki-ras a chromosome 12 gene, Fig. 1 (b)). This variable and regional lip gene amplification in DNA derived from normal kidney tissues adjacent to Wilms' tumour may be of great importance.
It has been argued that a spontaneous degree of overreplication does occur in non-tumourigenic normal cells (14). There is evidence that preferred chromosomal regions for amplification of genes exist and possibly 11 p 5 4 3 2 1 ' n w"n w"n w"b n w"b N w"   Fig. 2 (c) was re-hybridized with these probes (e.g.vVI catalase in Fig. 2 (f) Fig. 1. (a), (f): Autoradiograph of the Hind III filter re-hybridized with a catalase probe